The present invention is directed to a method for assessing the biological or pharmacological activity of a test material without the need to identify any of the constituents of that material, by exposing mammalian cells to the material and assessing whether structural changes, such as protein phosphorylation or protein-protein interaction, are induced in proteins present in the mammalian cells. While the present methods are useful for any test material, these methods have particular utility for testing materials which include complex mixtures of molecules where, for example, the physiological effects of the complex mixture may be a result of a synergy between two or more constituents present in the mixture. Thus, the present methods can be used to assess the biological activity of herbs, herbal extracts, plant extracts, animal extracts, natural or synthetic compounds, or combinations thereof. This method facilitates the formulation of natural products with consistent biological or pharmacological activities without the need to identify any of the chemical constituents responsible for the biological or pharmacological response.
Modern medical and pharmaceutical sciences typically attempt to treat disease by prescription of a single, highly purified and well-characterized pharmaceutical compound whose activity has been carefully measured so that a precise dosage may be administered. The use of such highly purified drugs facilitates the manufacture of uniform dosage forms because drug concentration can simply and accurately be used to predict the appropriate pharmacological dosage.
However, use of a single highly purified drug is not always appropriate nor desirable for treating disease. In some instances, nutrients and/or pharmacologically active compounds may act together, in synergy. For example, research indicates that several constituents in saw palmetto extracts operate in synchrony to inhibit proliferation of cells in benign prostatic hyperplasia (BHP).
The most frequent reason men consult a urologist is because of an impairment in urinary flow. In men over 45 years of age, the cause of impaired urinary flow is often benign prostatic hyperplasia (BHP). The cause of BHP is an abnormal, but nonmalignant, proliferation of cells and tissues within the prostate gland. Eventually, urethral obstruction leads to urinary retention, kidney damage, and infection. In advanced stages, surgical resection is the treatment of choice.
To understand how medications may affect BPH, medical researchers seek the mechanism(s) that are believed to cause the condition. In the prostate, testosterone from the blood is converted by an enzyme to the more potent androgen, dihydrotestosterone (DHT). DHT increases the expression of proteins with resultant changes in cell metabolism and proliferation. In the process of normal growth, sex accessory organs are relatively insensitive to testosterone and DHT after puberty. However, in hyperplastic prostatic tissues the concentrations of DHT may be four to six times those of normal prostatic tissue. Thus, researchers infer that these high concentrations of DHT result in increased growth of the gland in mature males. Drugs have been developed to reduce the effects of androgens, for example, estrogens. However, while estrogens do reduce the effect of androgens they cause feminization, impotence, and cardiovascular toxicity in menxe2x80x94side effects which are highly undesirable.
In addition to androgenic stimulation, infiltration of the prostate by inflammatory cells is an etiologic factor in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils, produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, derivatives of arachidonic acid have been extensively studied. Thus, medical research indicates that the best therapeutic regimen for BHP would address both androgenic and inflammatory mechanisms.
Several plants contain compounds with antiandrogenic and anti-inflammatory properties, for example, saw palmetto which consists of the partially dried, ripe fruit of Serenoa repens. Saw palmetto was recognized as a xe2x80x9cdrugxe2x80x9d in the United States from 1906 to 1950 and was once widely used for a variety of ailments, particularly those of the urogenital tract, until losing popularity in the United States after World War II. European scientists continued to study saw palmetto and recognized that, in patients suffering form BPH, an extract of the fruit produced increased urinary flow, reduced residual urine, increased ease in commencing micturition, and decreased frequency of urination.
While extensive clinical and laboratory studies have been undertaken and reported, the mechanism of action of saw palmetto is poorly understood. Studies have shown that a liposterolic extract of the berries reduced cellular uptake of both testosterone and DHT by more than 40 percent. This mechanism is confirmed by the observation that saw palmetto extract does not induce changes in the level of testosterone, or other hormones, in the plasma. Other studies have indicated that an extract of saw palmetto reduces the conversion of less active testosterone to the more active DHT by inhibiting the enzyme 5xcex1-reductase.
In addition to their antiandrogenic properties, saw palmetto berries may also have anti-inflammatory activity. This may to be due to the inhibition of the cyclooxygenase and 5-lipoxygenase pathways, thereby preventing the biosynthesis of inflammation-producing prostaglandins and leukotrienes. Together, the antiandrogenic and anti-inflammatory effects seem to account for the beneficial role of the herb in treating BPH. Placebo-controlled, double-blind clinical studies carried out on more than 2,000 BPH patients in Germany have confirmed the effectiveness of a saw palmetto extract in such conditions.
A large number of possibly active ingredients have been isolated from saw palmetto including large amounts of beta-sitosterol-3-D-glucoside. Anthranilic acid, caffeic acid, chorogenic acid, tannin, sugars, and polysaccharides, are also present. Unfortunately, the active antiandrogenic principles remain unidentified, although they are known to reside in the acidic lipophilic fraction of the berries. The inability to identify a single, active compound indicates that a combination of ingredients may be responsible for the beneficial activities of saw palmetto. Alteration of the combination through purification of single ingredients results in a concomitant loss of the original biological activity.
However, the pharmaceutical industry generally relies upon purifying and quantifying an active ingredient in order to standardize preparation of medications.
The present invention provides a solution to this problem by providing methods for assessing the biological activity of a test material without the need for identifying and quantifying the active constituents of the test material. According to the present invention, cellular health and function can be assessed by observing the type and amount of certain proteins in the cell. Thus, the methods of the present invention expose mammalian cells to a test material and assess whether new proteins are synthesized by the cell or structural changes are induced in cellular proteins.
Cellular function is a direct result of both transcriptional and translational control processes. It has long been recognized that transcriptional control is necessary to differentiate one cell type from another. However, in addition to the variety of transcriptional controls developed by cells, cellular function is also dependent on the type and amount of post-translational modification of cellular proteins. One such post-translational modification is protein phosphorylation. Phosphorylation of serine, threonine and tyrosine residues on proteins is a fundamental post-translational regulatory process for mediating signal transduction, gene transcription, RNA splicing, cellular adhesion, apoptosis and cell cycle control. Wipf et al., 5 BIOORGANIC and MEDICINAL CHEMISTRY 165 (1997). According to the present invention, the type and extent of structural changes in cellular proteins is a measure of the physiological state of a cell. Thus, the cell reacts to environmental stimuli by increasing or decreasing protein phosphorylation, and by reorganizing how proteins interact with each other.
Specific, individual compounds are known to increase protein phosphorylation. For example, fluoride has been shown to enhance protein tyrosine phosphorylation of various constituents in the intracellular signaling cascade of osteoblasts. Ammann et al., 22 BONE 39 (1998). Ethanol and cocaine have been shown to increase the phosphorylation rate of certain phosphoproteins. Torres and Horowitz, 20 PROG. NEURO-PSYCHOPHARMACOL. and BIOL. PSYCHIAT. 561 (1996). Lead may induce phosphorylation in brain cells. Markovac et al., 96 TOXICOL. and APP. PHARM. 14 (1988). Dioxin may induce protein phosphorylation in trout. Newstad et al., 119 TOXICOL. and APP. PHARM. 41 (1993).
Some methods for detecting or identifying certain types of molecules have been developed which utilize phosphorylated proteins. For example, U.S. Pat. No. 5,496,703 to Babish et al., issued Mar. 5, 1996, discloses methods for detecting dioxins by exposing mammalian cells to a test sample which may contain such dioxins, examining a cell lysate of the mammalian cells for the presence of phosphorylated proteins and correlating the phosphorylation level of those phosphorylated cells with a standard set of phosphorylated proteins derived from mammalian cells known to be exposed to dioxin or dioxin-like molecules. U.S. Pat. No. 5,695,944 to Croce et al., issued Dec. 9, 1997, is directed to a method of identifying compounds that modulate bcl-2 mediated cell death by contacting a cell with a test compound and detecting whether the bcl-2 in the cell is phosphorylated at a higher or lower level than a control cell which is not contacted with the test compound. U.S. Pat. No. 5,672,470 to Hengstenberg et al., issued Sep. 30, 1997, is drawn to a method for detecting toxic substances, by incubating a sample with a suspension of bacteria which have a phosphotransferase system so that a substrate analog is phosphorylated and then hydrolyzed by an enzyme in the phosphotransferase system. U.S. Pat. No. 5,618,677 to Ni et al., issued Apr. 8, 1997, is directed to a method of testing whether a compound modulates a phosphate cotransporter protein by exposing a cell with the human inorganic phosphate cotransporter protein to a test compound and measuring a change in inorganic phosphate uptake relative to a control cell which is not exposed to the test compound. U.S. Pat. No. 4,871,661 to Webb et al., issued Oct. 3, 1989, is directed to a method of screening materials for carcinogenic properties by administering those materials to test animals and observing whether the animal produces a 60 kilodalton cancer-associated phosphoprotein.
While the prior art discloses that protein tyrosine kinases, which phosphorylate proteins, play a fundamental role in signal transduction pathways and provides procedures for detecting phosphorylated proteins, these references do not disclose that complex mixtures of molecules are capable of producing reproducible signaling patterns of phosphoproteins, or that mammalian phosphorylation patterns may accordingly be used to assess the total biological activity of a complex mixture of molecules.
The present invention is directed to a method for determining whether a test material has biological activity which includes, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing the tested mammalian cells, and comparing the pattern of phosphorylated proteins in those tested mammalian cells to the pattern of phosphorylated proteins in control cells. The test material can be a mixture of molecules, an herb, a mixture of herbs, an herbal extract, a plant extract and the like. The control cells are either the same cultured mammalian cells which have not been exposed to the test material or the same cultured mammalian cells which are in a quiescent, or non-dividing condition. In a further embodiment, the pattern of phosphorylated proteins in the tested mammalian cells may be compared to the pattern of phosphorylated proteins in positive control cells. Such positive control cells are the cultured mammalian cells which have been exposed to a known beneficial and non-toxic compound, an FDA approved drug or a beneficial plant or herbal extract of proven efficacy.
The present invention is also directed to a method for determining whether a test material has biological activity which includes, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing the tested mammalian cells to produce a mixture of cellular proteins, electrophoretically-separating the cellular proteins, reacting the cellular proteins with a monoclonal antibody directed against a phosphorylated amino acid and comparing the pattern of phosphorylated proteins in the tested mammalian cells to the pattern of phosphorylated proteins in control cells. Again, the control cells are the same cultured mammalian cells which have not been exposed to the test material. The pattern of phosphorylated proteins in the these tested mammalian cells may also be compared to the pattern of phosphorylated proteins in positive control cells which have been exposed to a beneficial and non-toxic compound, an FDA approved drug or a beneficial plant or herbal extract of proven efficacy.
The present invention is further directed to a method for detecting synergy of biological activity between a first component and a second component in a complex mixture of components which comprises separating the complex mixture of components into a first fraction and a second fraction, and testing whether the first fraction induces the same pattern of protein phosphorylation as the complex mixture. In this instance, the first component is present in the first fraction and the second component is present in the second fraction.
The present invention is still further directed to a method for determining whether a first component has more biological activity than a second component of a complex mixture of components which includes separating the complex mixture of components into a first fraction and a second fraction, and testing whether the first fraction induces the same pattern of protein phosphorylation as the second fraction. Again, the first component is present in the first fraction and the second component is present in the second fraction.
The present invention is also directed to a method for determining whether a test material has biological activity which includes, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing the tested mammalian cells, and comparing the pattern of protein-protein interaction in the tested mammalian cells to the pattern of protein-protein interaction in control cells. The control cells are the same cultured mammalian cells which have not been exposed to the test material. The protein-protein interaction in the tested mammalian cells may also be compared to the pattern of protein-protein interaction in positive control cells. Such positive control cells can be cultured mammalian cells which have been exposed to a beneficial and non-toxic compound such as an FDA approved drug, a beneficial plant or herbal extract of proven efficacy or the like.
The present invention is further directed to a method for comparing the biological activity of a test material to a control material which includes, incubating the test material with cultured mammalian cells to produce tested mammalian cells, lysing the tested mammalian cells, and comparing the pattern of phosphorylated proteins in the tested mammalian cells to the pattern of phosphorylated proteins in control cells. In this embodiment the control cells are the same cultured mammalian cells which have been exposed to a control material and have not been exposed to the test material. The control material is a beneficial and non-toxic compound such as an FDA approved drug, a beneficial plant or herbal extract of proven efficacy, or the like.